We are working on genetic approaches that target vector mosquitoes to control transmission of malaria parasites. We are testing the hypothesis that the introduction into a population of vector mosquitoes of a gene conferring resistance to malaria parasites will decrease pathogen transmission and result in less disease and mortality In humans. The goals that must be met to test this hypothesis Include the development of mosquitoes engineered genetically to be incapable of the transmission of malaria parasites. Major steps in the laboratory efforts to develop these insects Included the adaptation of transgenesis technologies for the stable introduction of genes into the appropriate vector mosquito species, the discovery of promoter sequences from mosquito genes that could be used to express effector molecules at a time and place In the insects so as to optimize the impact on the target pathogens, and the formulation of effector genes that efficiently prevent the development of pathogens while at the same time Impose negligible fitness loads on the mosquitoes carrying them. The Specific Aims of this proposal address the working hypothesis that it should be possible to engineer the effector genes so as to achieve the target of zero parasite prevalence (percent of all mosquitoes with an infection) and mean intensities of infection (average number of parasites in only those mosquitoes infected) in the salivary glands of parasite-challenged mosquitoes. The Specific Aims are 1) optimize expression of single-chain antibodies (scFv) that disable Plasmodium falciparum In the midgut and hemolymph of transgenic Anopheles stephensi; 2) construct and test in parasite-challenge assays transgenic An. stephensi carrying single and multiple optimized scFvs for their ability to prevent parasites from infecting midguts and salivary glands; and 3) evaluate the fitness of strains of An. stephensi carrying one or more transgenes expressing anti-parasite scFvs relative to control laboratory colonies using life-table parameters. Gene cloning, transgenesis (both transposon- and site-specific recombination-mediated), fitness assays and parasite-challenge assays are used to develop and test genes that confer resistance In An. stephensi to the human malaria parasite, P. falciparum.